PROJECT SUMMARY/ABSTRACT Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cannot fully explain lung disease phenotype in cystic fibrosis (CF). Previous genome-wide association studies (GWAS) identified additional regions of the genome correlating with CF lung disease severity. One of these sites lies within the intergenic region between E47 like ETS transcription factor 5 (ELF5) and Ets-homologous factor (EHF), and Apaf-1 interacting protein (APIP) at chromosome 11p13. ELF5 is known to have significant roles in breast cancer and is thought to confer susceptibility to asthma in specific populations. EHF is a critical regulator of injury response and inflammation in the lung, and APIP has an important role in apoptosis. Our goal was to determine the functional significance of this region, which is predicted to be regulatory, since it lacks annotated genes. Cis- regulatory elements within the 11p13 modifier region were first identified in lung epithelial cells by mapping open chromatin using DNase I digestion and deep sequencing (DNase-seq). Next, histone modifications associated with active chromatin, H3K4me1 and H3K27ac, were revealed by chromatin immunoprecipitation. Predicted enhancer elements were assayed in bronchial epithelial cells using luciferase reporter genes. Two elements showed strong enhancer activity for the promoters of both EHF and ELF5. No enhancers of the APIP promoter were found. To determine direct physical interactions between cis-regulatory elements and gene promoters within 11p13, we used circular chromosome conformation capture and deep sequencing (4C-seq). 4C-seq confirmed the enhancer-promoter associations, identified additional interacting elements and defined CCCTC- binding factor (CTCF)-enriched boundaries of the topologically associated domains (TADs) at 11p13. No strong interactions were observed with the APIP promoter, which lies outside of the main TAD or sub-TAD encompassing the GWAS signal. These results focus attention on the likely role of EHF and ELF5 in modifying CF lung disease severity. We hypothesize that these epithelial selective genes are regulated by cis-elements found at 11p13, and that the dysregulation of these genes contributes to CF lung disease severity. We aim to test this hypothesis in two ways. First, we will use clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to remove or mutate cis-regulatory elements across the TAD in airway epithelial cells. Evaluation of transcription factor binding, histone modifications, gene expression and chromatin landscape in the modified cells will reveal key regulatory mechanisms at 11p13. Second, we will use VPR-mediated activation of promoters and cis-elements across 11p13 in a human bronchial epithelial cell line. This will reveal the functional impact of activating gene expression on key cellular processes such as migration, proliferation and apoptosis. In conjunction with these experiments, we will use conditionally reprogrammed primary human bronchial epithelial (HBE) cells derived from healthy donors as well as CF patients to assess the impact of VPR-activation on epithelial function and integrity using assays such as trans-epithelial resistance (TER) and scratch assays.